1. Field of the Invention
The present invention relates generally to the area of novel strategies for the improvement of chemotherapeutic intervention. In other aspects, the present invention provides novel methods and compositions that combine the potency of DNA damaging agents with the combined delivery of a tumor suppressor. The combination of DNA damaging factors with the heterologous expression of a tumor suppressor gene lead to a pronounced synergy over and above the actions of the individual components.
2. Description of Related Art
Current treatment methods for cancer, including radiation therapy, surgery, and chemotherapy, are known to have limited effectiveness. Lung cancer alone kills more than 140,000 people annually in the United States. Recently, age-adjusted mortality from lung cancer has surpassed that from breast cancer in women. Although implementation of smoking-reduction programs has decreased the prevalence of smoking, lung cancer mortality rates will remain high well into the 21st century. The rational development of new therapies for lung cancer will depend on an understanding of the biology of lung cancer at the molecular level.
It is now well established that a variety of cancers are caused, at least in part, by genetic abnormalities that result in either the over expression of one or more genes, or the expression of an abnormal or mutant gene or genes. For example, in many cases, the expression of oncogenes is known to result in the development of cancer. xe2x80x9cOncogenesxe2x80x9d are genetically altered genes whose mutated expression product somehow disrupts normal cellular function or control (Spandidos et al., 1989).
Most oncogenes studied to date have been found to be xe2x80x9cactivatedxe2x80x9d as the result of a mutation, often a point mutation, in the coding region of a normal cellular gene, i.e., a xe2x80x9cproto-oncogenexe2x80x9d, that results in amino acid substitutions in the expressed protein product. This altered expression product exhibits an abnormal biological function that takes part in the neoplastic process (Travali et al., 1990). The underlying mutations can arise by various means, such as by chemical mutagenesis or ionizing radiation. A number of oncogenes and oncogene families, including ras, myc, neu, raf, erb, src, fms, jun and abl, have now been identified and characterized to varying degrees (Travali et al., 1990; Bishop, 1987).
During normal cell growth, it is thought that growth-promoting proto-oncogenes are counterbalanced by growth-constraining tumor suppressor genes. Several factors may contribute to an imbalance in these two forces, leading to the neoplastic state. One such factor is mutations in tumor suppressor genes (Weinberg, 1991).
An important tumor suppressor gene is the gene encoding the cellular protein, p53, which is a 53 kD nuclear phosphoprotein that controls cell proliferation. Mutations to the p53 gene and allele loss on chromosome 17p, where this gene is located, are among the most frequent alterations identified in human malignancies. The p53 protein is highly conserved through evolution and is expressed in most normal tissues. Wild-type p53 has been shown to be involved in control of the cell cycle (Mercer, 1992), transcriptional regulation (Fields et al., 1990, and Mietz et al., 1992), DNA replication (Wilcock and Lane, 1991, and Bargonetti et al., 1991), and induction of apoptosis (Yonish-Rouach et al., 1991, and, Shaw et al., 1992).
Various mutant p53 alleles are known in which a single base substitution results in the synthesis of proteins that have quite different growth regulatory properties and, ultimately, lead to malignancies (Hollstein et al., 1991). In fact, the p53 gene has been found to be the most frequently mutated gene in common human cancers (Hollstein et al., 1991; Weinberg, 1991), and is particularly associated with those cancers linked to cigarettes smoke (Hollstein et al., 1991; Zakut-Houri et al., 1985). The overexpression of p53 in breast tumors has also been documented (Casey et al., 1991).
One of the most challenging aspects of gene therapy for cancer relates to utilization of tumor suppressor genes, such as p53. It has been reported that transfection of wild-type p53 into certain types of breast and lung cancer cells can restore growth suppression control in cell lines (Casey et al., 1991; Takahasi et al., 1992). Although DNA transfection is not a viable means for introducing DNA into patients"" cells, these results serve to demonstrate that supplying wild type p53 to cancer cells having a mutated p53 gene may be an effective treatment method if an improved means for delivering the p53 gene could be developed.
Gene delivery systems applicable to gene therapy for tumor suppression are currently being investigated and developed. Virus-based gene transfer vehicles are of particular interest because of the efficiency of viruses in infecting actual living cells, a process in which the viral genetic material itself is transferred. Some progress has been made in this regard as, for example, in the generation of retroviral vectors engineered to deliver a variety of genes. However, major problems are associated with using retroviral vectors for gene therapy since their infectivity depends on the availability of retroviral receptors on the target cells, they are difficult to concentrate and purify, and they only integrate efficiently into replicating cells.
Tumor cell resistance to chemotherapeutic drugs represents a major problem in clinical oncology. NSCLC accounts for at least 80% of the cases of lung cancer; patients with NSCLC are, however, generally unresponsive to chemotherapy (Doyle, 1993). One goal of current cancer research is to find ways to improve the efficacy of gene replacement therapy for cancer by investigating interaction between the gene product and chemotherapeutic drugs. The herpes simplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir (Culver, et al., 1992). The HS-tK gene product is an exogenous viral enzyme, whereas the wt-p53 protein is expressed in normal tissues, suggesting that the modulation of chemoresistance by alterations in wt-p53 expression might be an alternative approach using a pathway mediated by an endogenous genetic program.
An adenovirus system has potential advantages for gene delivery in vivo, such as ease of producing high titer virus, high infection efficiency, and infectivity for many types of cells. The stability and duration of expression of the introduced gene are still controversial, however. The increase in p53 levels in cells that are sensitive to chemotherapeutic drugs can occur within 6 hours after DNA-damaging stimuli (Fritsche, et al., 1993, Zhan, et al., 1993), although increased p53 DNA binding activity can be reversed over the course of 4 hours if the stimulus is removed (Tishler, et al., 1993) Therefore, a high level of p53 expression can be maintained even after cessation of drug exposure. The expression of wt-p53 protein by Ad-p53 peaks at postinfection day 3 (14-fold greater than endogenous wild type) and decreases to a low level by day 9 (Zhang, et al., 1993). This suggests that a transiently high level of wt-p53 expression is sufficient to initiate the cytotoxic program in the cancer cell.
p53 has an important role as a determinant of chemosensitivity in human lung cancer cells. A variety of treatment protocols, including surgery, chemotherapy, and radiotherapy, have been tried for human NSCLC, but the long-term survival rate remains unsatisfactory. What is needed is a combination therapy that is used alone or as an effective adjuvant treatment to prevent local recurrence following primary tumor resection or as a treatment that could be given by intralesional injections in drug-resistant primary, metastatic, or locally recurrent lung cancer. Compositions and methods are also needed to developed, explore and improve clinical applicability of novel compositions for the treatment of cancer. Furthermore these methods and compositions must prove their value in an in vivo setting.
The present invention addresses the need for improved therapeutic preparations for use in killing cells by combining the effects of a tumor suppressor gene or protein and a DNA damaging agent or factor. The present invention also provides compositions and methods, including those that use viral mediated gene transfer, to promote expression of a wild-type tumor suppressor gene, such as p53, in target cells and to deliver an agent or factor that induces DNA damage. The inventors surprisingly found that using the compositions disclosed herein, they were able to induce programmed cell death, also known as apoptosis, in a very significant number of target cells.
Using the present invention the inventors have demonstrated a remarkable effect in controlling cell growth and in particular, tumor cell growth. Tumor cell formation and growth, also known as xe2x80x9ctransformationxe2x80x9d, describes the formation and proliferation of cells that have lost their ability to control cellular division, that is, they are cancerous. It is envisioned that a number of different types of transformed cells are potential targets for the methods and compositions of the present invention, such as: sarcomas, melanomas, lymphomas, and a wide variety of solid tumors and the like. Although any tissue having malignant cell growth may be a target, lung and breast tissue are preferred targets. The present inventors disclose herein that a p53-expressing recombinant delivery vector was able to markedly reduce the growth rate of cells when used in conjunction with a DNA damaging agent.
The invention provides, in certain embodiments, methods and compositions for killing a cell or cells, such as a malignant cell or cells, by contacting or exposing a cell or population of cells with a p53 protein or gene and one or more DNA damaging agents in a combined amount effective to kill the cell(s). Cells that may be killed using the invention include, e.g., undesirable but benign cells, such as benign prostate hyperplasia cells or over-active thyroid cells; cells relating to autoimmune diseases, such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasia and the like. Although generally applicable to killing all undesirable cells, the invention has a particular utility in killing malignant cells. xe2x80x9cMalignant cellsxe2x80x9d are defined as cells that have lost the ability to control the cell division cycle, as leads to a xe2x80x9ctransformedxe2x80x9d or xe2x80x9ccancerousxe2x80x9d phenotype.
To kill cells, such as malignant or metastatic cells, using the methods and compositions of the present invention, one would generally contact a xe2x80x9ctargetxe2x80x9d cell with a p53 protein or gene and at least one DNA damaging agent in a combined amount effective to kill the cell. This process may involve contacting the cells with the p53 protein or gene and the DNA damaging agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the p53 protein or gene and the other includes the DNA damaging agent.
Naturally, it is also envisioned that the target cell may be first exposed to the DNA damaging agent(s) and then contacted with a p53 protein or gene, or vice versa. However, in embodiments where the DNA damaging factor and p53 are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the DNA damaging agent and p53 would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both agents within about 12-24 hours of each other, and more preferably within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred.
The terms xe2x80x9ccontactedxe2x80x9d and xe2x80x9cexposedxe2x80x9d, when applied to a cell, are used herein to describe the process by which a tumor suppressor gene or protein, such as p53, and a DNA damaging agent or factor are delivered to a target cell or are placed in direct juxtaposition with the target cell. TO achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell, i.e., to induce programmed cell death or apoptosis. The terms, xe2x80x9ckillingxe2x80x9d, xe2x80x9cprogrammed cell deathxe2x80x9d and xe2x80x9capoptosisxe2x80x9d are used interchangeably in the present text to describe a series of intracellular events that lead to target cell death. The process of cell death involves the activation of intracellular proteases and nucleases that lead to, for example, cell nucleus involution and nuclear DNA fragmentation. An understanding of the precise mechanisms by which various intracellular molecules interact to achieve cell death is not necessary for practicing the present invention.
DNA damaging agents or factors are defined herein as any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage, such as, xcex3-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as xe2x80x9cchemotherapeutic agentsxe2x80x9d, function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. In certain embodiments, the use of cisplatin in combination with a p53 protein or gene is particularly preferred as this compound.
Any method may also be used to contact a cell with a p53 protein, so long as the method results in increased levels of functional p53 protein within the cell. This includes both the direct delivery of a p53 protein to the cell and the delivery of a gene or DNA segment that encodes p53, which gene will direct the expression and production of p53 within the cell. In that protein delivery is subject to such drawbacks as protein degradation and low cellular uptake, it is contemplated that the use of a recombinant vector that expresses a p53 protein will provide particular advantages.
A wide variety of recombinant plasmids and vectors may be engineered to expresses a p53 protein and so used to deliver p53 to a cell. These include, for example, the use of naked DNA and p53 plasmids to directly transfer genetic material into a cell (Wolfe et al., 1990); formulations of p53-encoding DNA trapped in liposomes (Ledley et al., 1987) or in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983); and p53-encoding DNA coupled to a polylysine-glycoprotein carrier complex.
The use of recombinant viruses engineered to express p53 is also envisioned. A variety of viral vectors, such as retroviral vectors, herpes simplex virus (U.S. Pat. No. 5,288,641, incorporated herein by reference), cytomegalovirus, and the like may be employed, as described by Miller (Miller, 1992); as may recombinant adeno-associated virus (AAV vectors), such as those described by U.S. Pat. No. 5,139,941, incorporated herein by reference; and, particularly, recombinant adenoviral vectors. Techniques for preparing replication-defective infective viruses are well known in the art, as exemplified by Ghosh-Choudhury and Graham (1987); McGrory et al. (1988); and Gluzman et al. (1982), each incorporated herein by reference.
To kill a cell in accordance with the present invention, one would generally contact the cell with a p53 protein or gene and a DNA damaging agent in a combined amount effective to kill the cell. The term xe2x80x9cin a combined amount effective to kill the cellxe2x80x9d means that the amount of p53 and DNA damaging agents are sufficient so that, when combined within the cell, the cell is induced to undergo apoptosis. Although not required in all embodiments, the combined effective amount of p53 and DNA damaging agent will preferably be an amount that induces significantly more cell death than the use of either element alone, and most preferably, the combined effective amount will be an amount that induces synergistic cell death in comparison to the effects observed using either element alone.
A number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein, as well as the size of multicellular tumor spheroids formed, such as those colonies formed in tissue culture. In vitro cell killing is particularly shown in Example 7 of the present disclosure. Alternatively, one may measure parameters that are indicative of a cell that is undergoing programmed cell death, such as, the fragmentation of cellular genomic DNA into nucleosome size fragments, generally identified by separating the fragments by agarose gel electrophoresis, staining the DNA, and comparing the DNA to a DNA size ladder. Nucleosome size fragments are identified as a progressive steps or ladders of monomers and multimers having a base unit of about 200 basepairs.
Similarly, a xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount of a p53 protein or gene and DNA damaging agent that, when administered to an animal in combination, is effective to kill cells within the animal. This is particularly evidenced by the killing of cancer cells, such as lung, breast or colon cancer cell, within an animal or human subject that has a tumor. xe2x80x9cTherapeutically effective combinationsxe2x80x9d are thus generally combined amounts of p53 and DNA damaging agents that function to kill more cells than either element alone, and preferably, combined amounts that bring about a synergistic reduction in tumor burden.
Studying certain in vivo and ex vivo parameters of cell death are therefore also effective means by which to assess the effectiveness of the composition and methods of the invention. For example, observing effects on the inhibition of tumorigenicity, as measured by TdT expression of frozen tissue sections or by using other staining methods and target antigens, as known to skilled pathologists. Naturally, other means of determining tumor mass, growth, and viability may also be used to assess the killing of target cells. In particular, one may assess the effects in various animal model systems of cancer, including those in which human cancer cells are localized within the animal. Animal models of cancer, unlike those of AIDS, are known to be highly predictive of human treatment regimens (Roth et al., editors (1989)). One exemplary embodiment of a predictive animal model is that in which human small-cell lung cancer cells (H358 cells) are grown subcutaneously. Using this system, the inventors have shown that p53-bearing adenovirus instilled intratumorally, along with the co-administration of a chemotherapeutic agent, gives rise to a surprisingly effective tumor reduction.
A particularly preferred method of delivering a p53 protein to a cell is to contact the cell with a recombinant adenovirus virion or particle that includes a recombinant adenoviral vector comprising a p53 expression region positioned under the control of a promoter capable of directing the expression of p53 in the given cell type.
The p53 expression region in the vector may comprise a genomic sequence, but for simplicity, it is contemplated that one will generally prefer to employ a p53 cDNA sequence as these are readily available in the art and more easily manipulated. In addition to comprising a p53 expression unit and a promoter region, the vector will also generally comprise a polyadenylation signal, such as an SV40 early gene, or protamine gene, polyadenylation signal, or the like.
In preferred embodiments, it is contemplated that one will desire to position the p53 expression region under the control of a strong constitutive promoter such as a CMV promoter, viral LTR, RSV, or SV40 promoter, or a promoter associated with genes that are expressed at high levels in mammalian cells such as elongation factor-1 or actin promoters. All such variants are envisioned to be useful with the present invention. Currently, a particularly preferred promoter is the cytomegalovirus (CMV) IE promoter.
The p53 gene or cDNA may be introduced into a recombinant adenovirus in accordance with the invention simply by inserting or adding the p53 coding sequence into a viral genome. However, the preferred adenoviruses will be replication defective viruses in which a viral gene essential for replication and/or packaging has been deleted from the adenoviral vector construct, allowing the p53 expression region to be introduced in its place. Any gene, whether essential (e.g., E1A, E1B, E2 and E4) or non-essential (e.g., E3) for replication, may be deleted and replaced with p53. Particularly preferred are those vectors and virions in which the E1A and E1B regions of the adenovirus vector have been deleted and the p53 expression region introduced in their place, as exemplified by the genome structure of FIG. 1.
Techniques for preparing replication defective adenoviruses are well known in the art, as exemplified by Ghosh-Choudhury and Graham (1987); McGrory et al. (1988), and Gluzman et al., each incorporated herein by reference. It is also well known that various cell lines may be used to propagate recombinant adenoviruses, so long as they complement any replication defect which may be present. A preferred cell line is the human 293 cell line, but any other cell line that is permissive for replication, i.e., in the preferred case, which expresses E1A and E1B may be employed. Further, the cells can be propagated either on plastic dishes or in suspension culture, in order to obtain virus stocks thereof.
The invention is not limited to E1-lacking virus and E1-expressing cells alone. Indeed, other complementary combinations of viruses and host cells may be employed in connection with the present invention. Virus lacking functional E2 and E2-expressing cells may be used, as may virus lacking functional E4 and E4-expressing cells, and the like. Where a gene which is not essential for replication is deleted and replaced, such as, for example, the E3 gene, this defect will not need to be specifically complemented by the host cell.
Other than the requirement that the adenovirus vectors be engineered to express p53, the nature of the initial adenovirus is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention. This is because Adenovirus type 5 is a human adenovirus about which there is significant amount of biochemical and genetic information known, and which has historically been used for most constructions employing adenovirus as a vector.
The methods and compositions of the present invention are equally suitable for killing a cell or cells both in vitro and in vivo. When the cells to be killed are located within an animal, e.g., lung, breast or colon cancer cells or other cells bearing a p53 mutation, both the p53 protein or gene and the DNA damaging agent will be administered to the animal in a pharmacologically acceptable form. The term xe2x80x9ca pharmacologically acceptable formxe2x80x9d, as used herein, refers to both the form of any composition that may be administered to an animal, and also the form of contacting an animal with radiation, i.e., the manner in which an area of the animals body is irradiated, e.g., with irradiation, X-rays, let UV-irradiation, microwaves, electronic emissions, and the like. The use of DNA damaging radiation and waves is known to those skilled in the art of irradiation therapy.
The present invention also provides advantageous methods for treating cancer that, generally, comprise administering to an animal or human patient with cancer a therapeutically effective combination of a p53 protein or gene and a DNA damaging agent. This may be achieved using a recombinant virus, particularly an adenovirus, that carries a vector capable of expressing p53 in the cells of the tumor. The p53 gene delivering composition would generally be administered to the animal, often in close contact to the tumor, in the form of a pharmaceutically acceptable composition. Direct intralesional injection of a therapeutically effective amount of a p53 gene, such as housed within a recombinant virus, into a tumor site is one preferred method. However, other parenteral routes of administration, such as intravenous, percutaneous, endoscopic, or subcutaneous injection are also contemplated.
In treating cancer according to the invention one would contact the tumor cells with a DNA damaging agent in addition to the p53 protein or gene. This may be achieved by irradiating the localized tumor site with DNA damaging radiation such as X-rays, UV-light, xcex3-rays or even microwaves. Alternatively, the tumor cells may be contacted with the DNA damaging agent by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a DNA damaging compound, such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. The DNA damaging agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a p53 protein, gene or gene delivery system, as described above.
The surprising success of the present invention is evidenced by the finding that using Ad5CMV-p53 virus in combination with cisplatin yielded profound results in studies using a nude mouse model. The combined virus-DNA damage therapy regimen significantly inhibited the tumorigenicity of H358 cells, a cell that normally produces a significant tumor mass. The tumorigenicity of the lung cancer cells was inhibited through the treatment by Ad5CMV-p53, but not by the control virus expressing luciferase, indicating that the p53 protein in combination with a DNA-damaging agent has great therapeutic efficacy.
A number of methods for delivering chemotherapeutic formulations, including DNA expression constructs, into eukaryotic cells are known to those of skill in the art. In light of the present disclosure, the skilled artisan will be able to deliver both DNA damaging agents and p53 proteins or genes to cells in many different effective ways.
For in vivo delivery of DNA, the inventors envision the use of any gene delivery system, such as viral- and liposome-mediated transfection. As used herein, the term xe2x80x9ctransfectionxe2x80x9d, is used to describe the targeted delivery of DNA to eukaryotic cells using delivery systems, such as, adenoviral, AAV, retroviral, or plasmid delivery gene transfer methods. The specificity of viral gene delivery may be selected to preferentially direct the gene to a particular target cell, such as by using viruses that are able to infect particular cell types. Naturally, different viral host ranges will dictate the virus chosen for gene transfer, as well as the likely tumor suppressor gene to be expressed for killing a given malignant cell type.
It is also envisioned that one may provide the DNA damaging chemotherapeutic agent through a variety of means, such as by using parenteral delivery methods such as intravenous and subcutaneous injection, and the like. Such methods are known to those of skill in the art of drug delivery, and are further described herein in the sections regarding pharmaceutical preparations and treatment.
For in vitro gene delivery, a variety of methods may be employed, such as, e.g., calcium phosphate- or dextran sulfate-mediated transfection; electroporation; glass projectile targeting; and the like. These methods are known to those of skill in the art, with the exact compositions and execution being apparent in light of the present disclosure.
Other embodiments concern compositions, including pharmaceutical formulations, comprising a p53 protein or gene in combination with a DNA damaging agent, such as cisplatin. In such compositions, the p53 may be in the form a DNA segment, recombinant vector or recombinant virus that is capable of expressing a p53 protein in an animal cell. These compositions, including those comprising a recombinant viral gene delivery system, such as an adenovirus particle, may be formulated for in vivo administration by dispersion in a pharmacologically acceptable solution or buffer. Preferred pharmacologically acceptable solutions include neutral saline solutions buffered with phosphate, lactate, Tris, and the like.
Of course, in using viral delivery systems, one will desire to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the cell, animal or individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
Preferred pharmaceutical compositions of the invention are those that include, within a pharmacologically acceptable solution or buffer, a p53 protein, or more preferably a p53 gene, in combination with a chemotherapeutic DNA damaging agent. Exemplary chemotherapeutic agents are adriamycin, 5-fluorouracil, camptothecin, actinomycin-D, hydrogen peroxide, mitomycin C, cisplatin (CDDP), and etoposide (VP-16), with the use of cisplatin being particularly preferred.
Still further embodiments of the present invention are kits for use in killing cells, such as malignant cells, as may be formulated into therapeutic kits for use in cancer treatment. The kits of the invention will generally comprise, in suitable container means, a pharmaceutical formulation of a recombinant vector that is capable of expressing a p53 protein in an animal cell, and a pharmaceutical formulation of a DNA damaging agent. The recombinant vectors and DNA damaging agents may be present within a single container, or these components may be provided in distinct or separate container means. In a preferred embodiment, the recombinant vector will be a recombinant p53-expressing adenoviral vector present within an adenovirus particle and the DNA damaging agent will be cisplatin.
The components of the kit are preferably provided as a liquid solution, or as a dried powder. When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.